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Down was born in Torpoint, Cornwall. He was descended on his father’s side from an Irish family, his great-great grandfather having been Catholic bishop of Derry. His sister’s daughters eventually married into the Adrian, Darwin and Keynes families. John Down went to local schools including the Devonport Classical and Mathematical School. At 14 he was apprenticed to his father, the village apothecary at Anthony St Jacob’s. The vicar gave him a present of Arnott’s Physics which made him determined to take up a scientific career. At the age of 18 he went to London where he got a post working for a surgeon in the Whitechapel Road where he had to bleed patients, extract teeth, wash bottles and dispense drugs. Later he entered the pharmaceutical laboratory in Bloomsbury Square and won the prize for organic chemistry. He also met Michael Faraday and helped him with his work on gases. More than once he was called back to Torpoint to help his father in the business until he died in 1853. Down would have liked a career in science but in those days there were few prospects for a career in chemistry.

Instead, he decided to take up medicine, entering the Royal London Hospital as a student in 1853. There he had a career distinguished by honours and gold medals and he qualified in 1856 at the Apothecaries Hall and the Royal College of Surgeons. In 1858 he was appointed Medical Superintendent of the Earlswood Asylum for Idiots in Surrey. People were astonished that he should wish to pursue a career working in the neglected and despised field of idiocy. He had been one of the outstanding students of his time with every prospect of election to the staff of the London Hospital. He was concerned that all children who were afflicted by mental alienation or incapacity of any kind were placed in the category of idiots and regarded as beyond help. He had been enthusiastic about hearing of an experimental school in Switzerland but on visiting it found the inmates neglected and the Patron of the school living it up in the West End of London.

He decided to transform Earlswood which was a large institution which had its origins in two pioneering institutions set up in Highgate and Colchester. Meanwhile he took his MB in London, won the Gold Medal in physiology and took his MRCP and MD degrees. He was elected Assistant Physician to the London Hospital and continued to live at Earlswood and practice there and in London. John Langdon Down was quite liberal and advanced for a Victorian gentleman. He vigorously defended the higher education of women and denied that it made them more liable to produce feeble minded offspring. His ethnic classification of idiots led him to maintain that if a mentally defective member of a white race could show the racial features of a non-white race, it proved that racial differences were non-specific. He used this argument to refute the apologists for Negro slavery in the Southern States at the time of the American Civil War and to support the concept of the unity of mankind.

In 1866 he wrote a paper entitled "Observations on the Ethnic Classification of Idiots" in which he put forward the theory that it was possible to classify different types of conditions by ethnic characteristics. He listed several types including the Ethiopian type. He is most famous for his classification of what is known as Down syndrome, named after him, but which he classified as the Mongolian type of Idiot.

In 1868 John Down set up his own private home for the "mentally subnormal" at Normansfield, between Hampton Wick and Teddington. His sons Reginald and Percival both qualified in medicine at the London Hospital, joined their father and became responsible for it after his death in 1896. Reginald was particularly interested in "Mongolian defectives" and noticed characteristics of their palmar lines. He also questioned whether the characteristics were too superficial for them to be seen as Mongolian and suggested that it might be an instance of "variation". It is not known whether he was aware of the pioneering work on chromosomes being done by his contemporary, August Weismann.